Survey of pharmaceutical industry's best practices around in vitro transporter assessment and Implications for Drug Development: Considerations from the IQ Transporter Working Group.

The IQ Transporter Working Group had a rare opportunity to analyse a cross-pharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of pre-incubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of cross-transport inhibition (P-gp, BCRP, OATP1B and OCT1) with high molecular weight ({greater than or equal to}500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1 and MATE1 suggesting that prediction of DDIs for these transporters will be common. In contrast inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates whilst compounds with MW <500 Da tended to be OAT3 substrates. Interestingly the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whilst those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. Significance Statement A diverse dataset showed transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1 and MATE1 was frequent if the compound inhibited other transporters. In contrast inhibition of OAT1 did not correlate with the other drug transporters tested.

Serological and Molecular Detection of Toxoplasma gondii in Domestic Pigs Intended for Human Consumption and Potential Occupational Hazard to Pig Farmers in India.

Toxoplasma gondii, an important food-borne zoonotic parasite, poses a worldwide public health hazard. Domestic pigs are considered one of the main intermediate hosts in the zoonotic transmission of T. gondii. To date, seroepidemiological information on T. gondii in domestic pigs in India is very scarce, and there are no reports of occupational hazards to pig farmers in this country. Here, we aimed at estimating the occurrence of T. gondii (antibodies and parasite DNA) in slaughtered pigs and pig farmers in Central India. Seroprevalence was determined in 410 serum samples from slaughtered pigs and 103 sera from pig farmers using an in-house prepared antigen-based modified agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and indirect-fluorescent antibody test (IFAT). Anti-T. gondii IgG antibodies were detected in 200 pigs (up to 48.8%, confidence interval [95% CI]: 40.4-52.2) and 44 pig farmers (up to 42.7%, 95% CI: 35.6-47.3) using MAT, ELISA, and IFAT. Inter-rater agreement showed an excellent agreement (kappa κ = 0.9) among the different serological tests suggesting similar detection potential of these tests. Recently acquired infections in all seropositive subjects were determined using IgG avidity testing and polymerase chain reaction (PCR). IgG avidity showed that 20 (10.3%) of slaughtered pigs and 8 (19.5%) pig farmers had a recently acquired infection. PCR for B1 and 529 repeats was performed in the heart tissues of slaughtered pigs and the blood cells of pig farmers. T. gondii DNA was detected in 14 (7.2%) slaughtered pigs and 5 (12.2%) pig farmers. Univariate analysis revealed that adult animals (>1 year), cats and rodents on the farm, and outdoor access are common factors (p ≤ 0.05) associated with T. gondii infection in pigs. Our results indicate that T. gondii is widely distributed in slaughtered pigs and pig farmers at risk of infection, highlighting a potential zoonotic transmission and health risk to consumers.

Serological and molecular detection of Toxoplasma gondii and Neospora caninum in free-ranging rats from Nagpur, India.

Toxoplasma gondii and Neospora caninum are cyst-forming coccidian parasites that infect both wild and domestic non-felids as intermediate hosts, with rodents serving as important reservoir hosts during their life cycles. This study was aimed at investigating T. gondii and N. caninum infections and identifying factors favouring T. gondii infection in free-ranging rats from India. A total of 181 rodents were trap-captured, and blood and brain samples were subsequently collected for serological and molecular examination of T. gondii and N. caninum. Antibodies against T. gondii and N. caninum were detected by MAT/NAT and IFAT in 13.8% (25/181) and 1.65% (3/181) of rodents, respectively. All three N. caninum samples positive by NAT/IFAT were also positive for ELISA, while for T. gondii, 19 of 25 MAT/IFAT positive samples were also positive for ELISA. The antibody titers (MAT/NAT/IFAT) of rodents seropositive for T. gondii ranged from 25 to 400, while those of rats seropositive for N. caninum ranged from 25 to 100. Also, using PCR, DNA from T. gondii (B1 gene) and N. caninum (NC5 gene) was found in 2.76% (5/181) of brain samples and 0.55% (1/181) of brain samples. All PCR positive samples were also seropositive. No mixed infections were observed in the serological and molecular detections. A Chi-square analysis revealed that older rats and rats living in urban areas are significantly associated with T. gondii infection; however, rodent species, gender, location, habitat types, and seasonality were statistically nonsignificant. Overall, this study demonstrated that T. gondii was widely distributed while N. caninum was less prevalent among free-ranging rats in the studied area.

Mechanistic Static Model based Prediction of Transporter Substrate Drug-Drug Interactions Utilizing Atorvastatin and Rifampicin.

OBJECTIVE: An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. METHODS: In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. RESULTS: The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. CONCLUSIONS: This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.

Drug transporters in drug disposition - the year 2022 in review.

On behalf of all the authors, I am pleased to share our third annual review on drug transporter science with an emphasis on articles published and deemed influential in signifying drug transporters' role in drug disposition in the year 2022. As the drug transporter field is rapidly evolving several key findings were noted including promising endogenous biomarkers, rhythmic activity, IVIVE approaches in transporter-mediated clearance, new modality interaction, and transporter effect on gut microbiome. As identified previously (Chothe et Cal. 2021, 2022) the goal of this review is to highlight key findings without a comprehensive overview of each article and to this end, each coauthor independently selected 1-3 peer-reviewed articles published or available online in the year 2022 (Table 1). Each article is summarized in synopsis and commentary with unbiased viewpoints by each coauthor. We strongly encourage readers to consult original articles for specifics of the study. Finally, I would like to thank all coauthors for their continued support in writing this annual review on drug transporters and invite anyone interested in contributing to future versions of this review.

The role of Toxoplasma TFIIS-like protein in the early stages of mRNA transcription.

BACKGROUND: The mRNA transcription is a multistep process involving distinct sets of proteins associated with RNA polymerase II (RNAPII) through various stages. Recent studies have highlighted the role of RNAPII-associated proteins in facilitating the assembly of functional complexes in a crowded nuclear milieu. RNAPII dynamics and gene expression regulation have been primarily studied in model eukaryotes like yeasts and mammals and remain largely unchartered in protozoan parasites like Toxoplasma gondii, where considerable gene expression changes accompany stage differentiations. Here we report a key modulator of RNAPII activity, TFIIS in Toxoplasma gondii (TgTFIIS). METHODS: A Pull-down assay demonstrated that TgTFIIS binds to RNAPII subunit TgRPB1. Truncation mutants of TFIIS help us define the regions critical for its binding to TgRPB1. Co-immunoprecipitation analysis confirmed the interaction between the native TgTFIIS and TgRPB1. Confocal microscopy revealed a predominantly nuclear localization. Native TgTFIIS was able to bind promoter DNA which was consistent with the CHIP results. RESULTS: TgTFIIS complements initiation defects in yeast mutants, and the regions implicated in RNAPII binding appeared essential for this function. Interestingly, the C-terminal zinc finger domain necessary for its potential elongation function is dispensable for TgRPB1 binding. TgTFIIS was found to be associated with the promoter region along with its association with the ORF on an RNAPII transcribed gene. CONCLUSION: The observations were in line with the potential role of TgTFIIS in early events of RNAPII transcription in addition to elongation. GENERAL SIGNIFICANCE: The study elucidates the potential role of RNAPII-associated proteins in multiple steps of transcription.

Seroprevalence, risk factors, and serological cross-reactivity for diagnosis of Toxoplasma gondii and Neospora caninum infections in goats in India.

Toxoplasma gondii and Neospora caninum are genetically related cyst-forming protozoan parasites that cause reproductive failures in ruminants. Given the limited information on the epidemiology of these infections in goats in India, the study aimed to estimate the seroprevalence, assess antibody cross-reactivity for diagnosis, and identify associated risk factors. A total of 695 sera were evaluated for antibodies to T. gondii and N. caninum infections using Modified Agglutination Test (MAT for Toxoplasma)/Neospora agglutination test (NAT), Enzyme-linked immunosorbent assay (ELISA), and Indirect Fluorescent Antibody Test (IFAT for tachyzoite and bradyzoite stages). The seroprevalence rate of T. gondii and N. caninum infections was 56.9% and 10.9%, respectively. Inter-rater agreement (kappa value - κ) was calculated to assess agreements between various diagnostic assays, using the IFAT as the gold standard (for detecting both infections), the agreements for MAT/NAT (κ = 0.97) and the ELISA (κ = 0.95) were excellent. The acute infection among seropositive goats were determined using serological (IgG avidity test - measures the binding strength between IgG antibodies and parasite antigens) and molecular diagnoses (PCR for repetitive DNA sequences - Toxoplasma B1 gene: 131 bp and Neospora NC5 gene: 328 bp). Among seropositive goats ≥80% had high IgG avidity and <10% of animals had low IgG avidity antibodies for both infections. Most low IgG avidity goats were PCR positive for the TgB1 gene (94.4%) or Nc5 gene (85.7%). In the serological assays, we used different dilutions of test serum to rule out the cross-reactivity owing to similar antigenic makeup between these two parasites. When the serological cross-reactivity was analyzed using invasion assay at a serum titer of ≥200, more than 90% T. gondii positive sera showed host cell invasion of N. caninum and vice versa. Largely, the serological results indicate that cut-off serum dilution of ≥1:200 for ELISA and IFAT and ≥1:25 for MAT/NAT avoids serological cross-reactivity between T. gondii and N. caninum. Further, the Univariate and multivariate analyses showed that adult animals (>2 years), reservoir hosts, and extensive rearing systems are common risk factors for these infections. However, the history of abortion was identified as a significant risk factor for T. gondii infection. This study revealed that T. gondii and N. caninum infections are highly prevalent in this region and the use of an appropriate cut-off serum dilution is necessary to avoid cross-reactivity between these closely related parasites.

Novel insights in drug transporter sciences: the year 2021 in review.

On behalf of the team I am pleased to present the second annual 'novel insights into drug transporter sciences review' focused on peer-reviewed articles that were published in the year 2021. In compiling the articles for inclusion, preprints available in 2021 but officially published in 2022 were considered to be in scope. To support this review the contributing authors independently selected one or two articles that were thought to be impactful and of interest to the broader research community. A similar approach as published last year was adopted whereby key observations, methods and analysis of each paper is concisely summarized in the synopsis followed by a commentary highlighting the impact of the paper in understanding drug transporters' role in drug disposition. As the goal of this review is not to provide a comprehensive overview of each paper but rather highlight important findings that are well supported by the data, the reader is encouraged to consult the original articles for additional information. Further, and keeping in line with the goals of this review, it should be noted that all authors actively contributed by writing synopsis and commentary for individual papers and no attempt was made to standardize language or writing styles. In this way, the review article is reflective of not only the diversity of the articles but also that of the contributors. I extend my thanks to the authors for their continued support and also welcome Diane Ramsden and Pallabi Mitra as contributing authors for this issue (Table 1).[Table: see text].

Seroprevalence and associated risk factors of Toxoplasma gondii and Neospora caninum infections in cattle in Central India.

Toxoplasma gondii and Neospora caninum are closely related cyst-forming parasites identified as important causes of reproductive failures in ruminants. While these parasites have been reported worldwide, seroprevalence and associated risk factors for cattle infections have not been determined in India. A total of 576 serum samples of cattle were analyzed for antibodies to T. gondii and N. caninum using enzyme-linked immunosorbent assay (ELISA), modified/Neospora agglutination test (MAT/NAT), and an indirect fluorescent antibody test (IFAT-tachyzoite and bradyzoite). Additionally, general information about cattle, movement of cats and dogs, the menace of rodents, management, and reproductive disorders were assessed to identify the potential risk factors. Overall, 32.9% (190/576) serum samples reacted positively to T. gondii and 24.8% (143/576) to N. caninum. The performance of the diagnostic tests showed excellent agreement between IFAT and ELISA (kappa [κ] = 0.98) and between MAT/NAT and ELISA (κ = 0.97). Combining both infections on avidity test, 94% sera had high-IgG avidity, and 3% had low-IgG avidity antibodies, indicating chronic infection in the majority of the cases. The identified risk factors (p < 0.05) for exposure to T. gondii were: increasing age (Odds Ratio [OR]: 2.02), movement of cat (OR: 4.8) and rodents (OR: 1.57) in the farm; and for N. caninum: increasing age (OR: 1.6), movement of dogs in the farm (OR: 2.07), drinking pond water (OR: 1.64) and abortion (OR: 1.82). These findings revealed that T. gondii and N. caninum infections are widespread in the study area and suggest conducting nationwide epidemiological studies owing to their economic importance.

A functionally divergent transcription elongation factor 1-like protein in Toxoplasma gondii.

Zinc ribbons, one of the largest fold groups among zinc fingers, often include proteins involved in the transcription machinery. Here, we identify and characterize one such zinc ribbon-bearing protein in the apicomplexan parasite Toxoplasma gondii, annotated as putative transcription elongation factor 1 (ELF1), with predicted functions in transcription and chromatin maintenance. We show that this ELF1 homolog, referred to as T. gondii ELF1-like divergent (TgELD), is expressed in both tachyzoite and bradyzoite developmental stages. TgELD associates with the cytoskeleton in the tachyzoites, while it transiently becomes a part of the cyst wall in the early bradyzoites, followed by a cytosolic and peripheral localization in late bradyzoites. TgELD is phosphorylated by a casein kinase 2-like protein, which has potential implications for its localization and function in the parasite.

A new species and new record of the genus Platybaetis Mller-Liebenau, 1980 (Ephemeroptera: Baetidae) from Eastern Himalaya, India.

A new species of Platybaetis viz., P. selvai sp. nov. is described herein based on larval collections from Tangon stream in the state of Arunachal Pradesh in Eastern region of Indian Himalaya. It can be differentiated by the following combination of characters: (i) posterior margin of abdominal segments IX with rounded 'U shaped spines; (ii) anterolateral margin of gills IVII with minute setae; (iii) claw with 78 denticles; (iv) paracercus composed of 1011 segments; (v) hindwing pads reduced, small. Brief ecological notes are appended.

Seroprevalence and risk factors of Toxoplasma gondii infection among veterinary personnel and abattoir workers in Central India.

Toxoplasmosis, caused by the protozoan parasite Toxoplasma gondii, is an important zoonotic infection. Veterinary personnel and abattoir workers are considered to be at a high risk of T. gondii infection owing to their occupational exposure. However, the association of T. gondii infection with occupational exposure to animals has not been determined in India. Hence, we analysed 139 and 126 blood samples of veterinary personnel and abattoir workers, respectively, for anti-T. gondii antibodies using enzyme-linked immunosorbent assay (ELISA), modified agglutination test (MAT) and indirect fluorescent antibody test (IFAT). The association of seroprevalence with sociodemographic profiles, work activities and dietary habits was determined in the study population. MAT, ELISA and IFAT results demonstrated nearly 46%, 48% and 47% seropositivity, respectively. MAT (kappa = 0.924) and IFAT (kappa = 0.962) results showed good agreement with ELISA results. Of the ELISA positive samples, 46% was copositive for IgG antibody, 1.5% for IgM antibody and 1.5% for both IgG and IgM antibodies. High IgG avidity was observed only in IgG+ IgM- and IgG+ IgM+ samples and not in IgM+ IgG- samples, indicating chronic T. gondii infection in most of the cases. Furthermore, multivariate analysis revealed that T. gondii seropositivity was associated with age > 30 years (odds ration [OR] = 1.992), cat at home (OR = 1.991), not wearing gloves (OR = 1.886), not wearing safety glasses (OR = 1.985) and contact with soil (OR = 1.695). These findings support the presence of a potentially significant association between T. gondii seropositivity and occupational exposure to animals.

Toxoplasma gondii induces robust humoral immune response against cyst wall antigens in chronically infected animals and humans.

Toxoplasma gondii differentiation from proliferating tachyzoites into latent bradyzoites is central to pathogenesis and transmission. Strong humoral immune response has been reported against tachyzoite antigens, however, antibody-mediated response towards bradyzoite antigens is poorly characterized. This work aimed to study the humoral immune response towards bradyzoite and associated cyst wall antigens particularly CST1. The immunoreactivity of 404 goats, 88 sheep and 92 human sera to recombinant (CST1 and SRS9) and native proteins of encysted bradyzoite along with well-established tachyzoite antigens (SAG1 and GRA7) was determined using ELISA, Western blot and immunofluorescence analysis (IFA). ELISA results revealed nearly 50% of sera contain T. gondii specific antibodies. Results were further validated using Western blot and IFA. T. gondii positive sera predominantly recognized the cyst wall besides the known tachyzoite surface antigens. The presence of CST1 antibodies in seropositive samples were in line with the staining patterns which were consistent with CST localization. Notably, T. gondii IgM- IgG+ sera recognize the cyst wall whereas IgM + IgG-sera recognize tachyzoite antigens indicating acute infection consistent with presence of parasite DNA. The study demonstrates a strong humoral response against bradyzoite associated cyst wall antigens across naturally infected animals and humans. CST1 emerged as a key immunomodulatory antigen which may have direct implications for clinical immunodiagnostics.

Molecular chaperone function of stress inducible Hsp70 is critical for intracellular multiplication of Toxoplasma gondii.

Intracellular pathogens like Toxoplasma gondii often target proteins and pathways critical for host cell survival and stress response. Molecular chaperones encoded by the evolutionary conserved Heat shock proteins (Hsps) maintain proteostasis and are vital to cell survival following exposure to any form of stress. A key protein of this family is Hsp70, an ATP-driven molecular chaperone, which is stress inducible and often indiscernible in normal cells. Role of this protein with respect to intracellular survival and multiplication of protozoan parasite like T. gondii remains to be examined. We find that T. gondii infection upregulates expression of host Hsp70. Hsp70 selective inhibitor 2-phenylethynesulfonamide (PES) attenuates intracellular T. gondii multiplication. Biotinylated PES confirms selective interaction of this small molecule inhibitor with Hsp70. We show that PES acts by disrupting Hsp70 chaperone function which leads to dysregulation of host autophagy. Silencing of host Hsp70 underscores its importance for intracellular multiplication of T. gondii, however, attenuation achieved using PES is not completely attributable to host Hsp70 indicating the presence of other intracellular targets of PES in infected host cells. We find that PES is also able to target T. gondii Hsp70 homologue which was shown using PES binding assay. Detailed molecular docking analysis substantiates PES targeting of TgHsp70 in addition to host Hsp70. While establishing the importance of protein quality control in infection, this study brings to the fore a unique opportunity of dual targeting of host and parasite Hsp70 demonstrating how structural conservation of these proteins may be exploited for therapeutic design.

Characterization of Toxoplasma gondii Spt5 like transcription elongation factor.

Elongation has emerged as a highly regulated step in the multistage process of transcription. Control of gene expression mediated through transcription elongation remains an unexplored area of study in Toxoplasma gondii where the demands of complex lifecycle necessitate a regulated transcription program. Here, we elucidate the central role of Spt5 homolog in T. gondii mRNA transcription. We demonstrate that TgSpt5 functions in conjunction with a small zinc finger protein TgSpt4. TgSpt5 interacts with TgRpb1, the largest subunit of RNA polymerase II and associates with actively transcribed genes. Enrichment of TgSpt5 towards the 3' end of genes coinciding with P-Ser2 form of RNAPII, a marker of active elongation further underscores its pivotal role in transcription. TgSpt5 undergoes phosphorylation mediated through Toxoplasma Cdk9 homolog, TgCrk9, which appears crucial for its function. Inhibition of TgCrk9, which also regulates RNAPII by differential phosphorylation of its C terminal domain, results in loss of TgSpt5 enrichment at 3' sites of the genes and an overall repressive effect on parasite progression. TgSpt5 along with TgSpt4 could successfully complement the loss of function mutations in yeast counterparts emphasizing its functional significance. Together, the results highlight the possible role of TgSpt5 in transcript elongation regulated through phosphorylation by TgCrk9.

Role of tyrosine residue (Y213) in nuclear retention of PCNA1 in human malaria parasite Plasmodium falciparum.

Proliferating Cell Nuclear Antigen (PCNA) undergoes several post-translational modifications including phosphorylation leading to its regulation in mammalian and yeast systems. Plasmodium falciparum possesses two PCNAs (PCNA1 & PCNA2) with an edge of PfPCNA1 over PfPCNA2 for DNA replication. Recent phospho-proteome data report phosphorylation of S191 residue without its functional implication. In mammalian cells, phosphorylation of HsPCNA at Y211 stabilizes chromatin bound PCNA. We find tyrosine (but not S191) to be conserved in PfPCNAs and it is important for its nuclear localization and foci formation of PfPCNA1. Further, a Y213F mutation in PfPCNA1 leads to its functional loss both in yeast and parasite. We highlight the importance of evolutionarily conserved tyrosine in PCNA from parasite to mammal linked with DNA replication and cell proliferation.

Prediction and Quantification of Hepatic Transporter-Mediated Uptake of Pitavastatin Utilizing a Combination of the Relative Activity Factor Approach and Mechanistic Modeling.

Quantification of the fraction transported (ft) by a particular transporter will facilitate more robust estimations of transporter interactions. Using pitavastatin as a model uptake transporter substrate, we investigated the utility of the relative activity factor (RAF) approach and mechanistic modeling to estimate ft in hepatocytes. The transporters evaluated were organic anion-transporting polypeptides OATP1B1 and OATP1B3 and sodium-taurocholate cotransporting polypeptide. Transporter-expressing human embryonic kidney 293 cells and human hepatocytes were used for determining RAF values, which were then incorporated into the mechanistic model to simulate hepatocyte uptake of pitavastatin over time. There was excellent agreement between simulated and observed hepatocyte uptake of pitavastatin, indicating the suitability of this approach for translation of uptake from individual transporter-expressing cells to more holistic in vitro models. Subsequently, ft values were determined. The largest contributor to hepatocyte uptake of pitavastatin was OATP1B1, which correlates with what is known about the in vivo disposition of pitavastatin. The ft values were then used for evaluating in vitro-in vivo correlations of hepatic uptake inhibition with OATP inhibitors rifampicin and cyclosporine. Predictions were compared with previously reported plasma exposure changes of pitavastatin with these inhibitors. Although hepatic uptake inhibition of pitavastatin was 2-3-fold underpredicted, incorporation of scaling factors (SFs) into RAF values significantly improved the predictive ability. We propose that calibration of hepatocytes with standard transporter substrates and inhibitors would allow for determination of system-specific SFs, which could subsequently be used for refining predictions of clinical DDI potential for new chemical entities that undergo active hepatic uptake.

Cdk-related kinase 9 regulates RNA polymerase II mediated transcription in Toxoplasma gondii.

Cyclin-dependent kinases are an essential part of eukaryotic transcriptional machinery. In Apicomplexan parasites, the role and relevance of the kinases in the multistep process of transcription seeks more attention given the absence of full repertoire of canonical Cdks and cognate cyclin partners. In this study, we functionally characterize T. gondii Cdk-related kinase 9 (TgCrk9) showing maximal homology to eukaryotic Cdk9. An uncanonical cyclin, TgCyclin L, colocalizes with TgCrk9 in the parasite nucleus and co-immunoprecipitate, could activate the kinase in-vitro. We identify two threonines in conserved T-loop domain of TgCrk9 that are important for its activity. The activated TgCrk9 phosphorylates C-terminal domain (CTD) of TgRpb1, the largest subunit of RNA polymerase II highlighting its role in transcription. Selective chemical inhibition of TgCrk9 affected serine 2 phosphorylation in the heptapeptide repeats of TgRpb1-CTD towards 3' end of genes consistent with a possible role in transcription elongation. Interestingly, TgCrk9 kinase activity is regulated by the upstream TgCrk7 based CAK complex. TgCrk9 was found to functionally complement the role of its yeast counterpart Bur1 establishing its role as an important transcriptional kinase. In this study, we provide robust evidence that TgCrk9 is an important part of transcription machinery regulating gene expression in T. gondii.

Rho Protein: Roles and Mechanisms.

At the end of the multistep transcription process, the elongating RNA polymerase (RNAP) is dislodged from the DNA template either at specific DNA sequences, called the terminators, or by a nascent RNA-dependent helicase, Rho. In Escherichia coli, about half of the transcription events are terminated by the Rho protein. Rho utilizes its RNA-dependent ATPase activities to translocate along the mRNA and eventually dislodges the RNAP via an unknown mechanism. The transcription elongation factor NusG facilitates this termination process by directly interacting with Rho. In this review, we discuss current models describing the mechanism of action of this hexameric transcription terminator, its regulation by different cis and trans factors, and the effects of the termination process on physiological processes in bacterial cells, particularly E. coli and Salmonella enterica Typhimurium.

Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii.

Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5'-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis.